Automated apparatus and methods for particle analysis, such as hematological analysis, typically pump a sample containing the particles suspended in a dispersion through a particle analyzer which detects differences in electrical, optical, chemical or other characteristics of the particles, and generates signals having characteristics relating to the differences. The signals are in turn transmitted to a processor for determining the parameters of the particle distributions.
Automated hematological analysis typically involves mixing within a cuvette or other mixing vessel a whole blood sample with several reagent-mixture components, such as diluent, and frequently one or more membrane-modifying agents, to create a reagent mixture which facilitates cell discrimination and identification. The reagent mixture is then pumped from the cuvette through the particle analyzer which detects the size and/or "opacity" of the blood cells by electrical and/or optical differences. The blood cells are detected or counted for a period of time sufficient to gather data for analysis, and data points are then stored and analyzed in a processor. The data may then be displayed in the form of a two-dimensional or three-dimensional histogram.
For many years, cell and particle counting and measuring instruments have measured the electrical resistance of cells or other particle suspensions in order to probe their structure and composition and in turn identify and discriminate among the subpopulations within a sample suspension. The early Coulter Counter.TM. instruments, sold by Coulter Electronics, Inc., operated upon the well-known principle of particle and cell measurement whereby a sample suspension is passed through a sensing orifice, a low-level dc voltage is applied across the orifice, and the change in impedance caused by the passage of the particles through the electric field of the orifice is measured to indicate the apparent particle size or volume.
Later developed instruments have provided information relating not only to particle size, but also characteristics due to the composition and nature of the particle material. Accordingly, these instruments have the capability to distinguish between cells of identical size having different intracellular characteristics, or other particles of identical size made of different materials. U.S. Pat. No. 3,836,849 shows one such instrument which generates particle sensing fields within the sensing orifice by means of both a dc current source and a radio frequency ("rf") current source, and in turn generates two or more interrelated output signals from the passage of each particle through the sensing orifice. A typical cell is composed of a thin, non-conducting outer cell membrane surrounding a cytoplasm containing other cell structure, such as a nucleus. Accordingly, the outer cell membrane functions as an insulator with respect to the relatively low level dc field, but is shorted out and is electrically "invisible" at the rf frequency. Thus, while all of the low-level dc current goes around the cell, some of the rf current goes through it. Accordingly, the resistance parameter detected in response to the dc field is the low-frequency conductivity depending only on, and indicative of the relative volume of the cell. The resistance parameter detected in response to the rf field, on the other hand, is the high-frequency conductivity determined by the resistivity of the cell interior as well as the relative volume. In an attempt to analogize the phenomenon to optical analysis, the ease with which the rf current will pass through a particle has been called "electrical transparency" or simply "transparency" of the particle; whereas the ability of the particle to impede the rf current has been called its "opacity".
The '849 patent further recognizes that different types of particles may exhibit the same or substantially the same opacity, and therefore suggests chemical treatment of select particles in order to alter their opacity and thereby facilitate in distinguishing between such particles having the same opacity.
One of the drawbacks of this type of prior art instrument is that it requires both dc and rf current sources, and in turn requires processing and analysis of at least two interrelated signals in order to provide an indication of both cell size and interior cell structure (also referred to as "intracellular complexity"). Accordingly, the additional costs of the rf source and related signal-processing electronics can render these instruments relatively expensive in comparison to those requiring a dc source only.
U.S. Pat. Nos. 4,368,423 and 4,472,506 to Liburdy show an apparatus and method for determining cell membrane dielectric breakdown in order to establish lymphocyte tumor cell cytotoxicity and the presence of actual tumor cells. Liburdy defines "dielectric breakdown" as occurring at the breakdown or breakpoint voltage whereby the lymphocyte cell membrane has undergone dielectric collapse and cell electrical resistivity has been markedly reduced. Liburdy recognizes that the breakdown voltage is directly related to the fluidity of the cell membrane and that lymphocyte cells which are cytotoxic possess decreased cell membrane fluidity. Liburdy's apparatus and method are therefore directed to determining cell membrane dielectric breakdown in order to establish lymphocyte tumor cell cytotoxicity as well as the presence of tumor cells. Liburdy draws the cells through a port within an electric field exposure tube, and a variable voltage source/amplifier is used to incrementally increase the voltage applied across the port and to the cells passing therethrough. The voltage source is set to stop at select intervals between 0 and 100 volts as the lymphocytes are drawn through the tube, a frequency histogram is acquired at each voltage exposure and stored, and the average voltage pulse height is plotted against the applied voltage to determine the breakdown profile of the lymphocyte cells.
One of the drawbacks of Liburdy's approach is that it is difficult and costly to construct a system that will take repeated and accurate measurements at each of the voltage increments. In addition, the cells are continuously drawn through the orifice and therefore different groups of cells are tested at each voltage increment, thus further increasing the likelihood of obtaining inconsistent and/or inaccurate test results. Because the system is dynamic, the flow conditions may likewise not be the same at each voltage increment, and therefore it is likely that the system will fail to repeatedly subject the different cells to the same test conditions.
Accordingly, it is an object of the present invention to provide a method and apparatus for particle analysis and cell differentiation, such as hematological analysis, which overcomes the above-described drawbacks and disadvantages of the prior art.